The invention relates to recombinant genetic material for expression of the genes of the biotin metabolic pathway in enterobacteria, microorganisms which contain this recombinant genetic material, and the use of such microorganisms in a biotechnological method of producing biotin. The invention furthermore relates to a method of producing biotin which comprises conversion of dethiobiotin by means of biotin synthase in a cell-free system.
Biotin (vitamin H) is a vitamin which is important for humans and animals and whose deficiency may cause, for example, seborrhoea, dermatitis, loss of appetite and lassitude. Accordingly, biotin is a beneficial additive to human and animal foods.
The production of biotin by methods of synthetic organic chemistry is elaborate and costly. For this reason, increasing attention is being paid to biotechnological methods in which biotin can be synthesized with the aid of microorganisms from low-cost starting materials such as glucose.
Escherichia coli (E. coli) is a microorganism which is able to synthesize biotin starting from simple carbon sources such as glycerol or glucose (FIG. 1). The genes responsible for biosynthesis of biotin in E. coli are present in an operon which has already been cloned and comprises the five genes bioA, bioB, bioC, bioD and bioF (also called bio genes hereinafter) (Gupta et al., Gene 1:331-345; 1977). These genes are transcribed in two different directions by a promoter-operator region which is located between the bioA and bioB genes. Related to the conventional gene map, the bioB, bioF, bioC and bioD genes are on the right and the bioA gene is on the left of the promoter-operator region. The DNA to the left of the promoter-operator region comprises, downstream of the bioA gene, another gene which is called ORFI (ORF=open reading frame) which codes for a polypeptide having 158 amino acids and is transcribed together with the bioA gene (Otsuka et al., J. Biol. Chem., 263:19577-19585; 1988). The function of the latter gene is as yet unknown. Other strains from the family of enterobacteria, for example of the genus Salmonella or Citrobacter, have a biotin operon of E. coli-analogous structure (Shiuan and Campbell, Gene 67:203-211; 1988).
Biotechnological methods of producing biotin which are carried out using microorganisms which are transformed with the cloned biotin operon of E. coli have already been disclosed. These methods are carried out starting from glucose. EP-B-236 429 describes, for example, microorganisms which are transformed with the biotin operon of E. coli, with the host organisms being mutated in their biA/bioR gene.
EP-A-316 229 describes E. coli mutants which produce less acetate and have likewise been transformed with the cloned biotin operon.
EP-A-449 724 discloses microorganisms which have been transformed with the biotin operon and additionally have mutations which result in smaller glucose consumption.
EP-A-266 240 furthermore discloses the cloning of the genes responsible for biotin synthesis in Bacillus sphaericus and a method, based on this, for producing biotin. This method must, owing to the metabolism of Bacillus sphaericus, be carried out starting from costly pimelic acid.
The yields obtained in the biotechnological methods disclosed are, however, as yet unsatisfactory from economic points of view.